Sådhanå (2019)44:50  Indian Academy of Sciences

https://doi.org/10.1007/s12046-018-1023-7 Sadhana(0123456789().,-volV)FT3
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Role of biocementation to improve mechanical properties of mortar

SNIGDHA P BHUTANGE1,*, M V LATKAR1 and T CHAKRABARTI2
1
Civil Engineering Department, Visvesvaraya National Institute of Technology (VNIT), Nagpur 440010, India
2
Nagpur, India
e-mail: snigdhabhutange12@gmail.com; mv.latkar@gmail.com; tapan1249@gmail.com

MS received 16 February 2018; revised 8 August 2018; accepted 25 September 2018

Abstract. Biocementation or Microbially Induced Calcium Carbonate Precipitation (MICCP) is a naturally

occurring green technique which involves the deposition of calcium carbonate by peculiar action of ureolytic
bacteria, which improves the mechanical properties of cementitious materials. These bacteria require a protein
source, a vitamin source and a carbon source for optimum growth. Urea and calcium source are necessary to
carry out the ureolysis reaction to get CaCO3 depositions which fill the voids within the cementitious materials
thereby improving their mechanical properties. To make the process economical, it is necessary to find out
optimum concentrations of the various chemicals and ingredients used in the process. Present study aims at
finding out the best possible combination of urea and protein source to carry out the MICCP process suc-
cessfully. There was 22.44% increase in compressive strength and 16.62% reduction in the water absorption of
experimental mortar cubes at 28 days of curing with the optimum combination of urea and protein source
concentrations.

Keywords. Biocementation; MICCP; calcium carbonate; ureolysis.

1. Introduction particles and provide strength to the same as a whole

In this era most of the population is looking forward to
urbanization and the construction industry has gained a
supreme importance. Mortar is one of the largest and bulk
consumption items in construction industry as it is used as a
binding material for bricks, stones and other masonry units
due to its low cost and availability. Although mortar has
many advantages, it is vulnerable to deterioration, corrosion
and cracks and hence loses its strength over a period of time
when subjected to variable weathering actions. Remedia-
tion and repair of construction works is a very costly affair.
Various synthetic filler agents, epoxy, epoxy resins and
different commercial admixtures are being used for this
purpose. However, these remedies are immensely expen-
sive and in many cases are not eco-friendly, thus there is
need of some alternative green and clean technology for
saving enormous structures from damage [1–3].
It is a well-known fact that calcium carbonate is most
abundantly occurring mineral in nature. Various microor-
ganisms have tendency to deposit calcium carbonate as a
part of their metabolic activity. These deposits are found
out to be very good binders between loose substrate
T. Chakrabarti: Formerly MPCB, Civil Engineering Department,
Visvesvaraya National Institute of Technology (VNIT), Nagpur,
440010, India
*For correspondence
structure by occupying the voids in between substrate
particles. Many natural structures such as ant hills on land
and coral reefs in water are best examples of calcification
occurring at ambient conditions and providing them
sustainability.
Microbially induced calcium carbonate precipitation
(MICCP) is the utilization of this capability of microor-
ganisms for strengthening the construction materials
thereby saving a lot of money and resources in an eco-
friendly and sustainable manner [4, 5]. The technique of
MICCP has been found to have high potential to remediate
cracks in building materials in an environmental friendly
manner and also to protect and consolidate decayed con-
struction materials [6, 7].
Ammonium concentration, oxygen availability, miner-
alogy and soil particle size are part of the conditions that
may influence ureolytic activity of the bacteria that are
assessed [8]. Soil as a source of bacteria provided with
nutrient rich environment, urea and a source of calcium are
the main players of the process of biocementation [9].
Abo-El-Enein et al described that the incorporation of
the moderately alkalophilic aerobic Sporosarcina pasteurii
increase the compressive strength of cement mortar cubes
by about 33% at the age of 28th day of curing [10].
It has also been revealed in one study that the use of
Bacillus sp. CT-5 bacteria in cement mortar specimen led to
40% increase in the compressive strength as compared to
50 Page 2 of 8 Sådhanå (2019)44:50

that of mortar specimen made with tap water and also about
50% reduction in the porosity [11]. In another study done
by Achal et al it has been investigated that the lactose
mother liquor (LML) which is a waste from dairy industry
can be used to replace conventional standard nutrient media
used for the growth of calcifying bacteria. Thus this study
provides a more sustainable way of construction material
improvement which is as good as the conventional way
[12]. Charpe et al have used general soil bacteria (without
isolation) which proved to be as effective as isolated cul-
tures. The use of lentil seeds as a source of protein and
sugar as a source of carbon make the process more eco-
nomically viable [9].
In a study carried out by Sun-Gyu Choi, instead of using
calcium chloride or other calcium salts, soluble calcium
was produced by mixing eggshell with vinegar and used for
the MICCP process which was just as good as that using
calcium chloride [13].
The study carried out by Gomez et al extended an
attention to application of MICCP from small scale labo-
ratory tests to large scale on field construction. A large tank
of 1.7 m diameter containing 0.7 m3 of a poorly-graded
natural sand was treated with large cementation injections.
Results were found out to be satisfactory even in large scale
operation [14].
The following reactions given by Eq. (1) to (7) explain
the sequence of processes occurring in MICCP [15].
• Initially, in the presence of urease, urea gets hydro-
lyzed to form carbamate and ammonia
COðNH2 Þ2 þH2 O ! NH2 COOH þ NH3 ð1Þ
• Additional one mole of ammonia and carbonic acid are
formed due to hydrolyses of carbamate
NH2 COOH þ H2 O ! NH3 þ H2 CO3 ð2Þ
• These products subsequently form 1 mole of bicar-
bonate and 2 moles of ammonium and hydroxide ions,
respectively.
H2 CO3 ! Hþ þ HCO
3 ð3Þ
ions, to form CaCO3 precipitation at the surface of the
cell, which serves as a nucleation site [15].
Ca2þ þ Cell ! CellCa2þ ð6Þ
CellCa2þ þ CO2
3 ! CellCaCO3 # ð7Þ
Present study was done by using cost effective ingredi-
ents such as sugar as a source of carbon instead of D-glu-
cose and lentil seeds (Lens culinaris) powder as a source of
protein instead of expensive peptone. Lentil seeds contains
25 g proteins/100 g dry weight [16].
2. Materials and methods
2.1 Preparation of experimental solution
For the present study, rhizospheric soil was used as a source
of bacteria instead of pure culture to make the process cost
effective. The quantity of soil taken was 200 g/l of water.
Meat extract as a vitamin source and sugar as a carbon source
were added in amounts 3 g/l and 0.5 g/l, respectively.
Commercial D-glucose was replaced by sugar as a carbon
source to reduce the cost. As a protein source for bacteria,
commercially used peptone was replaced by lentil seed
powder as its cost is about 53 times less than peptone. Lentil
seed powder was added in variable amounts for different sets
of experiments to find out the optimum concentration. The
resulting mixture was then incubated for 24 h at 37C. After
incubation, the optical density of the resulting mixture was
measured at 600 nm in spectrophotometer, which was found
out to be 0.856. This value of optical density indicates suf-
ficient cell biomass in the experimental solution. The
supernatant of the experimental solution was then separated
from the soil. Urea and gypsum (as a calcium source) were
then added to the solution to carry out the hydrolysis of urea
by the microbial mass in the experimental solution to form
CaCO3 precipitation. The amount of gypsum (calcium sul-
fate) used was double the urea concentration as calculated
from the stoichiometry of the chemical reaction.

2NH3 þ 2H2 O ! 2NHþ

4 þ 2OH

ð4Þ
2.2 Preparation of control 1 solution
• The previous reactions give rise to a pH increase due to
Control 1 solution was prepared to study the effects of added
which the bicarbonate equilibrium is shifted and
chemicals and other ingredients on the mechanical properties
carbonate ions are formed.
of mortar in the absence of bacteria. This was made exactly
HCO þ þ
3 þ H þ 2NH4 þ 2OH
 like experimental solution but without adding soil.
þ
! CO2
3 þ 2NH4 þ 2H2 O ð5Þ

• As the cell wall of a bacterium is generally negatively

2.3 Measurement of pH
charged, bacteria attract cations including Ca2? from
the environment which get deposited on their cell Since pH is an important parameter that indicates different
surface. Subsequently the Ca2?ions react with CO32- stages in a chemical reaction, the pH of the experimental
Sådhanå (2019)44:50 Page 3 of 8 50

Table 1. pH of the experimental solution at various experimental Table 3. Various combinations of concentrations of lentil seed
stages. powder and urea employed in the study.

Experimental stages pH range Combinations Lentil seed powder g/l Urea g/l
Before incubation 6.8–7.4 Combination 1 5 1
After incubation 4.7–5.2 Combination 2 10 1
After adding urea and gypsum 8.5–9.5 Combination 3 5 2
Combination 4 10 2
Combination 5 5 4
solution was measured at different stages of experiment. Combination 6 10 4
The values of pH of the experimental solution at different
stages are as shown in table 1.
concentration of lentil seed powder as a protein source and
concentration of urea were kept variable to suggest best
2.4 Sand and cement optimal combination of their concentrations. For every
combination of concentration of urea and lentil seed pow-
For the present study, locally available natural river sand der, six cubes for each 3, 7 and 28 days were cast. Table 3
was used confirming to IS 383 (1970) [17] of zone II having shows six different combinations of lentil seed powder and
specific gravity of 2.60. Ordinary Portland cement (OPC) urea. Three cubes of every combination were used for
confirming to IS 12269 (2013) [18] was used. The chemical compressive strength test and rest of the three cubes of were
composition of OPC is given in table 2. used for water absorption test for curing period of 3, 7 and
28 days.

2.5 Specimen preparation

The standard ratio of cement to sand i.e., 1:3 was main-
tained; also water to cement ratio was maintained to 0.42
throughout the study. Control, control 1 and experimental
specimens of standard size 70.7 mm 9 70.7 mm 9
70.7 mm were cast as per IS 4031 (part 6) 1988 [19].
Cylinders of size 100 mm 9 200 mm were prepared for
rapid chloride permeability test (RCPT) as per ASTM
C1202 [20]. All specimens were compacted well in vibra-
tion machine. The specimens were demolded after 24 h of
cast and kept for curing.
2.7 Curing conditions
The control specimens were cured in water. Control 1 and
experimental specimens were cured in curing solution
composed of sugar 0.5 g/l, urea 1 g/l and gypsum 2 g/l at
room temperature i.e. 27 ± 2C for 3, 7 and 28 days. This
was done to ensure that sufficient substrate, calcium source
and carbon source are available for the bacteria to carry out
the calcium carbonate formation reactions. Table 4 shows
the compositions of curing solutions used for control 1 and
experimental specimens.

2.6 Design of experiment 3. Results

Control specimens were made up of cement, sand and
water. Control 1 specimens were made up of cement, sand
and control 1 solution. Experimental specimens were made
up of cement, sand and experimental solution. The
Table 2. Chemical components of ordinary Portland cement.
3.1 Compressive strength test
IS 4031 (part 6) 1988 [19] was followed for the compres-
sive strength testing of control, control 1 and experimental
specimens of all six combinations mentioned in table 3 at

Material Components % Table 4. Compositions of curing solutions used for control 1 and
Ordinary Portland Cement Silicon dioxide (SiO2) 18.89 experimental specimens.
Aluminum oxide (Al2O3) 6.35
Sl. Type of Sugar g/l in Urea g/l in Gypsum g/l
Ferric oxide (FeO3) 3.87
no. specimen water water in water
Calcium oxide (CaO) 62.23
Magnesium oxide (MgO) 2.30 1 Control 0 0 0
Potassium oxide (K2O) 0.22 specimens
Sodium dioxide (Na2O) 0.17 2 Control 1 0.5 1 2
Sulphur trioxide (SO3) 0.83 specimens
Loss of ignition (LOI) 4.21 3 Experimental 0.5 1 2
Total 99.07 specimens
50 Page 4 of 8 Sådhanå (2019)44:50

Table 5. Average compressive strength of control, control and experimental specimens and % increase in average compressive strength
of experimental specimens as compared to control specimens at 3rd, 7th and 28th day of curing.

Average compressive
Average compressive Average compressive strength of
strength of control strength of control 1 experimental
specimens (N/mm2) specimens (N/mm2) specimens (N/mm2) p value (As per % Increase in
or MPa or MPa or MPa ANOVA) compressive strength

3 7 28 3 7 28 3 7 28 3 7 28 3 7 28
Combinations Days Days Days Days Days Days Days Days Days Days Days Days Days Days Days
Combination 1 32.14 34.68 44.01 30.68 34.41 41.35 33.61 36.01 45.35 0.0034 0.0003 0.020 4.56 3.85 3.03
Combination 2 32.28 35.34 45.35 32.01 34.01 41.35 33.34 36.68 46.15 3.31 3.77 1.76
Combination 3 24.41 30.68 33.88 23.67 27.34 31.08 27.34 35.34 41.48 12.02 15.22 22.44
Combination 4 29.48 30.01 36.01 27.01 30.01 34.01 32.54 35.48 43.35 10.41 18.22 20.37
Combination 5 28.68 33.34 37.34 29.34 32.28 36.14 32.14 38.68 44.15 12.09 16.00 18.21
Combination 6 31.48 34.54 37.61 30.28 33.61 37.48 34.14 38.14 44.68 8.47 10.42 18.79

the curing age of 3, 7 and 28 days, respectively. Table 5
shows the average compressive strengths of control, control
1 and experimental specimens and % increase in average
compressive strength of experimental specimens as com-
pared to control specimens at the curing age of 3, 7 and
28 days, respectively for all the six combinations. The
combination 3 i.e., 2 g/l of urea and 5 g/l of lentil seeds
powder gave best results.
3.2 Water absorption test
Water absorption test was performed on all types of spec-
imens as per ASTM C140-11a [21]. The specimens, after
curing periods of 3, 7 and 28 days were drawn from
respective curing solutions and wiped with a dry cloth. The
saturated weights of mortar specimens were taken. The
specimens were then kept in oven at a temperature range of
105C to 110C for 24 h. After 24 h of oven drying, dry
weights of specimens were noted down. The water
absorption was found out from difference between initial
and final weights. Combination 3 was found to give best
results i.e., 11.77 %, 13.77% and 16.62% reduction at 3rd,
7th and 28th days of curing, respectively (refer table 6).
The increase in compressive strength and decrease in
water absorption in experimental specimens are due to
calcium carbonate precipitation. This is due to the action of
ureolytic bacteria which hydrolyse urea thus producing
carbonate ions. When gypsum (calcium sulfate) is added as
calcium source, calcium carbonate is formed which being
insoluble gets deposited in the pores of matrix of mortar
specimens. This, in turn, causes increase in compressive
strength and decrease in water absorption (refer to chemical
reactions given in section 1). This is clear in the SEM
images presented in figure 2. It was also observed that there
was not much difference in the compressive strength and
water absorption of control and control 1 specimens. This
indicates that the chemicals and ingredients added other
than bacteria do not have any effect on the mechanical
properties of the mortar.
3.3 Statistical analysis
The data for compressive strength and water absorption was
analyzed by Analysis of Variance (ANOVA). The p value
for all the sets of experiments was found to be less than

Table 6. Percent reduction in water absorption at 3rd, 7th and 28th day of curing.

% Water absorption % Water absorption

% Water absorption of control 1 of experimental p value (As per % Reduction in water
of control specimens specimens specimens ANOVA) absorption

3 7 28 3 7 28 3 7 28 3 7 28 3 7 28
Combinations Days Days Days Days Days Days Days Days Days Days Days Days Days Days Days
Combination 1 5.78 5.88 4.83 5.76 5.83 4.82 5.75 5.88 4.74 0.0008 0.003 0.0001 0.50 0.00 1.87
Combination 2 5.88 5.83 4.89 5.87 5.75 4.82 5.80 5.75 4.87 1.26 1.38 0.37
Combination 3 5.85 5.70 4.88 5.58 5.29 4.65 5.16 4.92 4.07 11.77 13.77 16.62
Combination 4 5.83 5.61 4.80 5.57 5.42 4.59 5.20 4.86 4.06 10.87 13.44 15.50
Combination 5 5.87 5.63 4.86 5.40 5.06 4.75 5.25 5.00 3.98 10.53 11.11 18.15
Combination 6 5.78 5.88 4.83 5.63 5.07 4.19 5.26 5.00 3.99 9.03 14.89 17.36
Sådhanå (2019)44:50 Page 5 of 8 50

0.05. This shows that there is a significant variation

amongst the samples (see tables 5 and 6).

3.4 Durability tests

3.4a Chloride, sulfate and carbonation tests: Since com-
bination 3 was found out to be the best combination,
chloride, sulfate and carbonation tests were conducted for
all types of mortar specimens made by using combination
3. According to IS 456; 2000 allowable chloride content for
mortar containing non-embedded metal is 3 kg/m3 [22].
The chloride concentration was found out in extracted
experimental sample by Argentometric method and was
0.02 kg/m3 which is well within the limit. The sulfate
concentration of the extracted experimental sample was
obtained by turbidimetric method according to IS 3025
(part 24) 1986 [23]. The soluble sulfate concentration of the
experimental sample was 15.76 mg/l. This is also within
the limit, i.e., 400 mg/l as per IS 456:2000. For the car-
bonation test, phenolphthalein solution was sprayed on a
freshly exposed surface of the control and experimental
specimens [24]. A pink colour was obtained in control as
well as experimental specimens indicating that sufficient
Ca(OH)2 is present (refer to figure 1). This indicates that
both the specimens were not much carbonated. These tests
results indicate that the mortar prepared using experimental
solution is durable and resistant to weathering and does not
cause additional carbonation due to MICCP reaction
3.4b Rapid chloride permeability test (RCPT): Rapid Figure 2. SEM images of (a) control specimen and (b) experi-
chloride permeability test for best combination i.e., com- mental specimen.
bination 3 was performed to determine ability of mortar
specimens to resist the chloride ion penetration and their
control specimens was low and for experimental specimens
electrical conductivities. Rapid chloride permeability test
was very low according to ASTM C1202.
was performed according to ASTM C1202 (ASTM C1202,
2017) [20]. Control and experimental specimens were tes-
ted for RCPT. The average values of charge passed for
3.5 Scanning Electron Microscopy (SEM) analysis
control and experimental specimens were found out to be
1220 C and 458 C, respectively. The permeability class for To study the effect of biocementation, the 28 days speci-
mens of both control and experimental were examined

Figure 1. Carbonation test on (a) control specimen and (b) experimental specimen using phenolphthalein.
50 Page 6 of 8
Figure 3. EDX analysis of (a) control specimen and (b) experimental specimen.
under scanning electron microscope (JSM- 6380A). On
comparison of the SEM images of control and experimental
specimens, it was observed that the void spaces seen in the
control specimen were not seen in the experimental speci-
men. The Scanning Electron Micrographs are shown in
figure 2.
3.6 Energy dispersive X-ray (EDX) analysis
EDX analysis was done to confirm the presence of calcium
carbonate mineral in the matrix of both control and
experimental specimens. Figure 3a, b show the results of
EDX analysis for control and experimental specimens,
respectively. The amount of calcium is clearly more in
experimental specimen than in control specimen which may
be because of CaCO3 deposition by bacteria in experi-
mental specimen.
3.7 X-ray diffraction (XRD) analysis
The control and experimental specimens were analyzed by
XRD to find out the type of crystalline forms (vaterite,
Sådhanå (2019)44:50
aragonite and calcite) of the calcium carbonate crystals.
Using an XPERT-PRO diffractometer system, the XRD-
spectra were obtained with Cu anode (40 kV and 45 mA).
The specimens were crushed to an average particle size of
less than 10 microns. The XRD patterns of both specimens
were obtained as shown in figure 4a, b, by scanning spec-
imens by 2h value of 10 to 99 degrees. The experimental
specimen showed more number of peaks of vaterite, arag-
onite and calcite crystals than control specimen. This may
be due to calcium carbonate formation in experimental
specimen’s matrix due to MICCP reaction by ureolytic
bacteria.
4. Discussion
This study aimed to find out optimum concentrations of the
two crucially important ingredients i.e., urea and protein
source to carry out the MICCP reaction successfully by
using low cost ingredients as mentioned in section 2.1.
Table 5 shows the percent increase in compressive strength
of experimental specimens in comparison with control
specimens for all six combinations of concentrations of
Sådhanå (2019)44:50
Figure 4. XRD graphs of (a) control specimen and (b) experi-
mental specimen.
urea and lentil seeds powder. The results for water
absorption are depicted in table 6. The results show that
combination 3 having 2 g/l of urea and 5 g/l of lentil seeds
powder gave best improvement in compressive strength as
well as in water absorption than other higher concentra-
tions. Combination 3 showed 12.02%, 15.22% and 22.44%
increase in compressive strength and 11.77 %, 13.77% and
16.62% reduction in water absorption on 3rd, 7th and
28th days of curing, respectively. But when an attempt was
made to further minimize the concentration of urea to 1 g/l,
both the combination 1 and combination 2 showed no
significant change in compressive strength and water
absorption. This shows that urea as a substrate was not
sufficient in concentration below 2 g/l (combinations 1 and
2) for carrying out successful MICCP reaction. As far as
combinations 4, 5 and 6 are concerned, though there is a
significant improvement in mechanical properties, the
concentrations of lentil seed powder and urea are higher as
compared to combination 3. Hence it can be inferred that
combination 3 seems to be economically best amongst all
these combinations.
All these findings are supported by the results of SEM,
EDX and XRD tests. Durability tests were performed on the
experimental sample and the results of these tests showed
that the experimental mortar sample is durable and resistant
to weathering.
Page 7 of 8 50
5. Conclusion
In this era, where there is a rapid increase in the demand of
construction materials, MICCP or biocementation has been
proved to be most promising and novel technique to
maintain sustainability in an ecofriendly manner. Various
studies are underway to make use of biocementation to
strengthen structures and increase their durability. From the
present study, it can be concluded that biocementation
using experimental solution having 2 g/l of urea, and 5 g/l
of lentil seed powder is better for strength enhancement as
compared to other combinations of various concentrations
tested. Future research is needed to replace meat extract by
some other vitamin source to further reduce the cost of
experimental solution. Also, studies with different calcium
sources (other than gypsum) can be done.
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[24] RILEM, Measurement of hardened concrete carbonation
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